Project Summary The development and responsiveness of the mammalian immune system is shaped by a dynamic interaction between the host immune system and the resident microbiota. The development of a host of human diseases is associated with shifts in the microbiota alongside alterations in the intestinal immune responses. We previously established that a population of mononuclear phagocytes (MNPs) expressing the CX3CR1 chemokine receptor play a central role in the immune system's responses to the microbiota and regulation of intestinal homeostasis. In the presence of the microbiota, CX3CR1+ MNPs limit inflammatory immune responses against both pathogenic bacterial infections and the microbiota itself in addition to promoting Treg responses against soluble fed proteins. In contrast, removal of the microbiota promotes inflammatory T cell responses brought about by dysregulation in a number of CX3CR1+ MNP functions, including production of the anti-inflammatory cytokine IL-10 and clearance of intracellular bacteria. Further dysregulation of CX3CR1+ MNP function is associated with pathology in inflammatory conditions such as inflammatory bowel disease (IBD). We therefore hypothesize that under normal conditions, specific members of the microbiota activate cellular pathways in CX3CR1+ MNPs responsible for limiting intestinal inflammation by restraining inflammatory T cell responses and promoting intestinal mucosal barrier function. Conversely, the shifted microbiota composition frequently associated with gastrointestinal infection or inflammatory disorders such as IBD provide alternative signals to CX3CR1+ MNPs, driving them to pathogen clearance and promoting intestinal inflammation. Reestablishing the normal microbiota should then direct CX3CR1+ MNPs to restore intestinal homeostasis. To address the in vivo role for CX3CR1+ MNPs in the induction of intestinal immune responses, we generated novel mouse strains in which CX3CR1+ MNPs can be selectively depleted. We will use these mice in conjunction with ex vivo assays to study the regulation of homeostatic functions in CX3CR1+ MNPs by the microbiota as a whole as well as by select individual members. In Aim 1, we will determine how the microbiota modulates CX3CR1+ MNP function to promote regulatory T cell responses and limit inflammation. In Aim 2, we will determine the impact of individual members of the microbiota and their gene products on CX3CR1+ MNP- induced anti-inflammatory T cell responses. At the conclusion of these studies, we will have identified and characterized cellular pathways regulated by the microbiota that are designed to limit inflammation as well as the microbial gene products responsible for the induction of these anti-inflammatory pathways. Together, these studies will provide critical insights into the modulation of anti-inflammatory responses in the intestine by the microbiota and will facilitate the identification of additional clinical targets for promoting and reestablishing intestinal homeostasis.